Figure 1.

Zfp238 Interacts with Foxo1 and Inhibits Its Activity

(A and B) Interaction between exogenous Zfp238 and Foxo1. HEK293 cells were co-transfected with cMyc-tagged WTFoxo1 or CNFoxo1 and FLAG-tagged Zfp238 and cultured in the presence of serum. At 48 h after transfection, cells were harvested and lysates were immunoprecipitated (IP) with anti-FLAG (A) or anti-cMyc (B) or normal mouse IgG and blotted with anti-cMyc (A) or anti-FLAG (B).

(C) Immunofluorescence of transfected cMyc-WTFoxo1 and FLAG-Zfp238 in HEK293 cells.

(D) Overexpression of Zfp238 inhibits Foxo1-induced 5XGAL4-luciferase activity. At 36 h after co-transfection with pTAL-5XGAL4, phRL-SV40, PM-WTFoxo1, and FLAG-Zfp238 or p3xFlag-CMV empty vector, HEK293 cells were harvested and luciferase activity was measured. An asterisk indicates a statistically significant difference between luciferase activity in the absence and presence of FLAG-Zfp238 (*p < 0.05 by one-way ANOVA). Data represent the mean ± SEM from three independent experiments.

(E) Knockdown of Zfp238 increases Foxo1-induced 5XGAL4-luciferase activity. An asterisk indicates a statistically significant difference between luciferase activity in the absence and presence of shRNA-6 Zfp238 (*p < 0.05 by one-way ANOVA). Data represent the mean ± SEM from three independent experiments.

(F) Knockdown of Zfp238 induces expression of Foxo1-target genes in differentiated 3T3-L1 cells in the absence of serum. At 10–12 days after induction of differentiation of 3T3-L1 cells infected with pSIREN-RetroQ encoding shRNA-6 Zfp238 or shRNA-SCR, cells were harvested and real-time PCR was performed. An asterisk indicates a statistically significant difference (*p < 0.05 by one-way ANOVA). Data represent the mean ± SEM from three independent experiments.